Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level

Abstract

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T‐lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high‐throughput approaches of three‐dimensional (3D) co‐cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin‐based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass‐spectrometry‐based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin‐like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)‐expressing GCT cells and mCherry‐expressing TM cells were co‐cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re‐analyses of secretome data with regard to lysine‐ and proline‐hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV‐ or fibronectin‐coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro‐tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin‐based chemotherapy for GCT patients.

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options

“…Together with our previous observations of certain molecular analogies between YST and HCC, we identified novel therapeutic targets whose inhibition resulted in apoptosis induction and / or cell cycle arrest in a drug-dependent manner in YST cells, while fibroblasts remained mostly unaffected, thereby opening a therapeutic window for the treatment of (cisplatin-resistant) YST patients. Overall, several phase 2 studies showed only limited clinical benefit from treatment with either the PARP1 / 2 inhibitor Olaparib (Giorgi et al 2020 ) and Veliparib (Mego et al 2021 ), c-Met inhibitor Tivantinib (Feldman et al 2013 ), ABL1 / 2 / KIT / PDGFRA / B inhibitor Imatinib (Einhorn et al 2006 ; Piulats et al 2007 ), BRAF / FGFR1 / KIT / PDGFRB / RAF1 inhibitor Sorafenib (Skoneczna et al 2014 ), VEGFR1-3 / PDGFR / FGFR / KIT / c-Fms inhibitor Pazopanib (Necchi et al 2017 ), mTOR inhibitor Everolimus (Fenner et al 2018 ), or VEGFR2 / PDGFRB inhibitor Sunitinib (Feldman et al 2010 ; Oechsle et al 2011 ; Reckova et al 2012 ) in heavily pretreated refractory GCT patients. Nevertheless, most of these studies did not stratify between GCT subtypes, so that no conclusion can be taken with regard to the efficacy for YST patients.…”

“…The used (tumor) cell lines were cultured as described previously and summarized in Additional file 5 : Table S1A (Skowron et al 2021b ; Burmeister et al 2022 ). Polarization of THP-1 cells was described earlier and was based on Genin et al ( 2015 ), Skowron et al ( 2022 ). XTT cell viability assays upon treatment with inhibitors (Additional file 5 : Table S1B) were performed as described previously (Skowron et al 2022 ).…”

“…Polarization of THP-1 cells was described earlier and was based on Genin et al ( 2015 ), Skowron et al ( 2022 ). XTT cell viability assays upon treatment with inhibitors (Additional file 5 : Table S1B) were performed as described previously (Skowron et al 2022 ). The human phospho-kinase arrays (R&D Systems via Bio-Techne, Wiesbaden, Germany) were carried out according to the manufacturer’s protocol and evaluated using the ‘Protein Array Analyzer’-Plugin for ‘Image J’ ( https://imagej.nih.gov/ij/ ) (Schneider et al 2012 ; Carpentier 2022 ).…”

CD24 targeting with NK‐CAR immunotherapy in testis, prostate, renal and (luminal‐type) bladder cancer and identification of direct CD24 interaction partners

“…Establishment of CD24-deficient EC cell lines (DCD24) via CRISPR/Cas9 technology was performed as reported previously [4,17,18]. For 3D cultivation in hanging drops, similar to our previous studies [19,20], 3 9 10 3 tumour cells per drop (40 lL) were seeded onto an inverted lid of a 15-cm cell culture dish before being inverted back onto the bottom chamber filled with phosphate-buffered saline for humidity. The next day, NK-92 or NK-92-CD24-CAR cells were added in the same manner at a ratio of 1 : 3 (9 x 10 3 ) in a volume of 10 lL per drop, supplemented with recombinant human interleukin-2 (Peprotech via Biozol, Eching, Germany), with a final concentration per drop of 5 ngÁmL À1 .…”

Immune checkpoint inhibitors and Chimeric Antigen Receptor (CAR)-T cell therapy: Potential treatment options against Testicular Germ Cell Tumors

“…Other soluble factors, in particular IL-8, can lead to an increase in NF-kB and ABCB1, which are responsible of reduced Cisplatin sensitivity: this mechanism, described in gastric cancer cells, could be applicable to GCTs as well ( 71 ). It is therefore interesting to note that precisely the destruction of tumor cells mediated by platinum-based chemotherapy could at the same time stimulate the secretion of protumoral factors in the tumor stroma ( 72 ).…”